Brocaenols A-C: Novel Polyketides from a Marine-Derived Penicillium brocae Tim S. Bugni,† Valerie S. Bernan,‡ Michael Greenstein,‡ Jeffrey E. Janso,‡ William M. Maiese,‡ Charles L. Mayne,§ and Chris M. Ireland*,† Departments of Medicinal Chemistry and Chemistry, University of Utah, Salt Lake City, Utah 84112, and Wyeth Research, Pearl River, New York 10965
[email protected] Received September 10, 2002
Abstract: Chemical investigation of a Penicillium brocae, obtained from a tissue sample of a Fijian Zyzyya sp. sponge, yielded two known diketopiperazines and three novel cytotoxic polyketides, brocaenols A-C. The brocaenols contain an unusual enolized oxepine lactone ring system that to the best of our knowledge is unprecedented in the literature. The structures were elucidated by using 2D-NMR methods including an INADEQUATE experiment. The absolute stereochemistry of brocaenol A was established by using a modified Mosher method. The taxonomy of the producing fungus was elucidated by using both morphological and rDNA sequence analysis.
Marine microorganisms have proven to be rich sources of secondary metabolites that have both unique structures1 and potential as pharmaceutical leads.2 In particular, fungi obtained from marine invertebrates have yielded novel metabolites with potent antibacterial and anticancer activities.3 This potential has led us to investigate cultured filamentous marine-derived fungi for antibacterial activity and cytotoxic activity against HCT116 human colon carcinoma cell lines. One isolate (F97S76) was obtained from a Fijian Zyzzya sp. sponge and was investigated due to its remarkable activity against both methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. Although the antibiotic activity was traced to the epicorazine family of diketopiperazines,4 the extract yielded three novel polyketides, brocaenols A-C (1-3), that showed moderate activity against an HCT116 human colon carcinoma cell line. The structure elucidation including absolute stereochemistry, the biological activity, and the taxonomy of the producing organism are presented here. * To whom correspondence should be addressed. Phone: (801) 581 8305. Fax: (801) 585 6208. † Department of Medicinal Chemistry. ‡ Wyeth Research. § Department of Chemistry. (1) Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1-48. (2) Fenical, W. Trends Biotechnol. 1997, 15, 339-341. (3) Jensen, P. R.; Fenical, W. In Drugs From the Sea; Fusetani, N., Eds.; Karger: Basel, 2000; p 6-29. (4) Deffieux, G.; Filleau, M. J.; Baute, R. J. Antibiot. 1978, 31, 11061109. Deffieux, G.; Baute, M. A.; Baute, R.; Filleau, M. J. J. Antibiot. 1978, 31, 1102-1105.
Brocaenol A (1) was isolated as a pale yellow amorphous solid with a molecular formula of C16H15O8 for the [M + H]+, established by HRFABMS. The 1H NMR spectrum showed eight signals: two methyls, one oxygenated methine, one olefinic methine, three aromatic methines, and one hydrogen-bonded phenol. Brocaenol A (1) formed a diacetate upon treatment with pyridine and acetic anhydride, and the 1H NMR spectrum showed that the diacetate still contained one exchangeable proton (δ 4.47). The trisubstituted benzene ring was established by using 1H, 13C, COSY, and HMBC NMR data. The 13C shifts of the aromatic carbons were consistent with a 1,3 oxygen substitution and an aryl ketone as shown in substructure A, Figure 1. Additionally, the downfield shift of the phenolic proton (δ 11.93) indicated that the phenol was hydrogen bonded, further supporting the aryl ketone. Another substructure, assigned by using COSY and HMBC data, contained an allylic alcohol and is shown as substructure B in Figure 1. COSY correlations showed that the oxygenated methine (H-5) was coupled to both the H-11 methyl and the H-7 olefinic proton. A COSY correlation between the H-11 methyl and the H-7 olefinic proton further supported the spin system assignment. An HMBC correlation between H-5 and the C-12 carbonyl indicated the position of the ester carbonyl. Although the two substructures were elucidated, connecting the two proved difficult. In an attempt to fully elucidate the carbon backbone of brocaenol A (1), a 2-D INADEQUATE NMR experiment was performed by using a standard pulse sequence. The data were analyzed with NMR Analyst software.5 With the exception of two carbons (C-13 and C-8), the entire backbone (Figure 2) was assembled from the INADEQUATE spectrum. Since C-13 is a methoxyl, no correlations were expected, but a correlation was expected for C-8. However, the intensity of this response would be reduced, since neither of the two carbons are protonated and little or no NOE would be expected. In addition the C-8/C-8a coupling constant would be expected to be unusually high since 1JC-C values increase with both increasing s-character and increasing electronegativity of bonded atoms. An approximate increase of 10 Hz for each oxygen would be expected and in this case would result in a coupling constant of 80 Hz or higher.6 Since the INADEQUATE experiment was optimized for a 1JC-C value of 55 Hz, the standard coupling constant observed in benzene, it is not surprising that the C-8/C-8a correlation was not observed. Lack (5) NMR Analyst; ScienceSoft, LLC: Salt Lake City, UT 84102. (6) Wehrli, F. W.; Marchand, A. P.; Wehrli, S. Interpretation of carbon-13 NMR spectra; John Wiley & Sons Ltd.: New York, 1988. 10.1021/jo020597w CCC: $25.00 © 2003 American Chemical Society
2014
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Published on Web 02/04/2003
FIGURE 1. Partial structures for compound 1 showing COSY correlations.
FIGURE 3. Model used to assign the configuration at C-5 with ∆δRS values in ppm.
of attached protons also means that C-8 and C-8a have long T1 relaxation times. Measured values were 11.4 and 10.6 s, which could also hinder the signal intensity. The position of C-8, the position of the oxygens, and the formation of the tricyclic ring system were elucidated by careful analysis of the 13C and HMBC data. The ether linkage between C-4a and C-10a was supported by the 13C chemical shifts of both carbons. Formation of the oxepine ring to complete the structure for brocaenol A (1) was supported by the 13C chemical shifts of C-9, C-8a, C-8, and the C-11 methyl. First, the relatively high-field shift of the aryl ketone (δ 185.2) required an adjacent olefin.7 Second, the shifts of C-8a (δ 103.5) and C-8 (δ 162.9) were consistent with a β-oxygenated enone system.8 Last, the high-field chemical shift of the C-11 (δ 12.9) methyl supported C-7 oxygenation. These data indicated a seven-membered enol ether ring as shown for compound 1. HMBC correlations from both C-8 and C-8a to H-7 further supported the ether bridge. The absolute stereochemistry of brocaenol A (1) was determined by using a modified Mosher method.9 Brocaenol A (1) was esterified with R- and S-methoxy(trifluoromethyl)phenylacetyl (MTPA) chloride in pyridine to form the corresponding S- and R-MTPA esters, respectively. The ∆δRS values were measured for H-7, H-11, and H-13 yielding +0.05, +0.17, and -0.17, respectively. On the basis of the model shown9 in Figure 3, the configuration at C-5 was assigned as R. The stereochemistry at position C-10a was confirmed by using a gHSQMBC10 and CD. By measuring the 3JC-H between H-5 and C-12, the stereochemsitry at position C-10a could be determined. The measured value (5.2 Hz)
was compared to the coupling constants calculated for each of the two possible diastereomers. Coupling constants were calculated by using a modified Karplus equation11 in which the dihedral angles were determined by molecular modeling. With use of CS Chem 3D Pro, conformations of each diastereomer were analyzed after energy minimization, using MM2 followed by semiempirical methods (AM1). When OH-5 and C-12 were syn, the dihedral angle was -108.2° and gave a coupling constant of 1.7 Hz. When OH-5 and C-12 were anti, the dihedral angle was -12.1° and gave a coupling constant of 6.5 Hz. Therefore, the relationship between OH-5 and C-12 was determined to be anti resulting in an Sconfiguration at C-10a. These results were also consistent with the CD. Brocaenol A (1) showed a negative Cotton effect at 304 nm, indicating S-configuration at C-10a based on CD spectra for secalonic acids12 and nidulalin A,13 both of which show a negative Cotton effect for an S-configuration. Brocaenol B (2) was isolated as a white crystalline solid. A molecular formula of C17H14O8 was established by HRCIMS. On the basis of the molecular formula and the 1H NMR spectrum, brocaenol B (2) differed from 1 by an additional O-methyl and a loss of two protons. The 13C and HMBC spectra indicated that brocaenol B (2) contained a ketone at position 5. Specifically, the downfield shift of C-7 as compared to 1 and correlations from H-7 and H-11 to a carbon at δ 197.2 (C-5) supported the presence of a ketone at position 5. An HMBC correlation between the methoxyl (C-15) at δ 4.06 and the carbon at δ 164.9 supported the methyl ether and its connectivity to the oxepine ring. Additional support for methylation of the enol came from the subsequent upfield shift of C-8a. On the basis of phenols8 and NMR chemical shift prediction,14 O-alkylation at position 8 would have a downfield effect on C-8 and an upfield effect on C-8a. The S-configuration at C-10a in 2 was determined by comparison of the CD spectrum to that of brocaenol A (1). Brocaenol C (3) was isolated as an amorphous pale solid. HRCIMS gave a molecular formula of C16H12O8. From the 1H NMR data, it was clear that brocaenol C (3) was closely related to brocaenol B (2) as the chemical shifts were nearly identical (Table 2). On the basis of the molecular formula, brocaenol C (3) differed from brocaenol B (2) by one methyl and differed from brocaenol A
(7) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH: New York, 1990; p 144. (8) Pretsch, E.; Buhlmann, P.; Affolter, C. Structure determination of organic compounds: Tables of spectral data, 3rd ed.; Springer: Berlin, Heidelburg, Germany, 2000. (9) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry 2001, 12, 2915-2925. (10) Williamson, R. T.; Marquez, B. L.; Gerwick, W. H.; Kover, K. E. Magn. Reson. Chem. 2000, 38, 265-273.
(11) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy; VCH: New York, 1990; p 143. (12) Anderson, R.; Buchi, G.; Kobbe, B.; Demain, A. L. J. Org. Chem. 1977, 42, 352-353. Cole, R. J.; Cox, R. H. In Handbook of toxic fungal metabolites; Academic Press: New York, 1981; pp 646-669. (13) Kawahara, N.; Sekita, S.; Satake, M.; Udagawa, S.; Kawai, K. Chem. Pharm. Bull. 1994, 42, 1720-1723. (14) GmbH, U. S., 1.1 ed.; Scientific Software Engineering: Hergiswil, 1999-2000.
FIGURE 2. Carbon skeleton from INADEQUATE correlations with
13C
assignments and coupling constants in italics.
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TABLE 1. NMR Data for Brocaenol A (1) in CDCl3 position
13C
1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 1-OH
160.3 112.0 135.7 106.5 153.0 71.7 127.9 137.0 162.9 103.5 185.2 108.4 76.4 12.9 172.0 53.4
a
1H
HMBCa
COSY
6.76 (dd, 8.1, 0.8) 7.49 (t, 8.1) 6.82 (dd, 8.1, 0.8)
3, 4 2, 4 2, 3
1, 4, 9a 1, 4a 2, 4a, 9, 9a
4.94 (dd, 1.9, 0.7)
7, 11
8a, 6, 7, 10a, 11, 12
6.53 (dq, 1.9)
5, 11
5, 6, 8, 11
1.71 (dd, 1.9, 0.7)
5, 7
4, 5, 6, 7, 8
3.78 (s) 11.93 (s)
12 1, 2, 9a
Numbers refer to the carbon.
TABLE 2. NMR Data for Compounds 2 and 3 compd 2a δC 1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 15 OH
160.8 113.1 134.8 105.9 152.5 197.2 113.8 172.8 164.9 96.8 181.9 108.5 85.1 5.8 165.0 53.8 57.0
δH
compd 3b HMBCc
δC
159.9 6.82 (d, 8.6) 1, 4, 9a 109.6 7.46 (t, 8.6) 132.4 6.81 (d, 8.6) 2, 4a, 9a 106.1 154.5 200.6 113.8 8.10 (q, 1.2) 5, 6, 10a 174.1 165.4 110.3 186.1d 107.2 89.5 1.78 (d, 1.2) 5, 6, 7 4.8 167.9 3.81 (s) 12 52.8 4.06 (s) 8 12.19 (s) 1, 2, 9a
δH 6.51 (d, 8.4) 7.27 (t, 8.4) 6.59 (d, 8.4)
HMBC 4 1, 4a 2, 4a
8.25 (bd, 1.3) 5, 6, 10a
1.73 (d, 1.3)
5, 6, 7
3.72 (s)
12
a Collected in CDCl . b Collected in MeOH-d . c Numbers refer 3 6 to the carbon. d Shift is from the 13C spectrum collected in DMSOd6.
(1) by two protons. On the basis of these data, brocaenol C (3) was proposed to be the oxidized analogue of 1. Assigning brocaenol C (3) proved problematic because the aryl ketone signal (C-9) was not observed in CD3OD. Interestingly, this signal was observed at δ 186.1 in DMSO; however, now the C-5 carbonyl was missing. Nonetheless, an HMBC correlation was observed from the C-5 carbonyl to H-11 at δC 198.6. The difficulty in observing all 16 carbon signals could be due to either tautomerization or a combination of long T1 relaxation times and insufficient material. The C-10a stereochemistry for compound 3 was determined to be S on the basis of the optical rotation. All three brocaenols (1-3) showed a positive rotation. The CD spectrum of brocaenol C (3) was not interpreted due to its complexity. The spectrum showed continuous oscillation with the highest intensity between 275 and 350 nm and is most likely due to tautomerization or rapid conformation conversion. Conversion of compound 1 to compound 3 was attempted to help confirm the structure 2016 J. Org. Chem., Vol. 68, No. 5, 2003
of brocaenol C (3), but attempts with PCC and TPAP/ NMO failed. All three compounds were tested in an HCT-116 cell line with use of the MTT assay and showed moderate cytotoxicity. The IC50 values for 1, 2, and 3 were 20, 50, and >50 µg/mL, respectively. Taxonomic characterization of the fungus was based on both morphology and sequence analysis of the ITS regions. Comparison with the personal collection of Stephen Peterson (National Center for Agricultural Utilization Research, US Department of Agriculture) revealed that the ITS region of F97S76 was identical with a newly described species Penicillium brocae (GenBank accession AF484396).15 Additional details for the taxonomic characterization can be found in the Supporting Information. This is the first reported chemical investigation of the recently described Penicillium brocae. Biosynthetically the brocaenols resemble terrestrial fungal metabolites such as the nidulalins13 and the secalonic acids.12 However, enolized oxepine lactones appear to be uncommon in natural products. Experimental Section General Experimental Procedures. Optical rotations were measured in a 5-cm cell in MeOH. All other general experimental details are the same as those reported in Bugni et al.16 Biological Material. Strain F97S76 was obtained from a tissue homogenate of a Zyzzya sp. sponge collected in Fiji. Strain F97S76 has been deposited in the Wyeth-Ayerst Culture Collection in Pearl River, NY. Fermentation, Extraction, and Isolation. Culture F97S76 grown on Bennett’s agar (10 g/L of dextrose, 1 g/L of beef extract [Difco], 1 g/L of yeast extract [Difco], 2 g/L of N-Z-Amine A [Quest International], 15 g/L of agar) was inoculated into 10 mL of potato dextrose broth (PDB, Difco), pH 7.0, in a 25 × 150 mm test tube and incubated at 22 °C, 160 rpm for 7 days. A second stage seed was prepared by transferring the 10 mL culture to a 250 mL Erlenmeyer flask containing 50 mL of PDB, which was incubated at 22 °C, 200 rpm for 4 days. Production fermentation was performed in a 2.8 L Fernbach flask containing 1 L of Czapek-Dox medium (Difco). Each flask was inoculated with 50 mL of second stage seed and incubated at 22 °C, 200 rpm for 7 days. One liter of ethyl acetate was added followed by shaking at 200 rpm for 1 h. The aqueous and ethyl acetate phases were separated and the ethyl acetate removed by rotary evaporation. The ethyl acetate extract (3.0 g) was dissolved in 110 mL of 10% H2O/MeOH and extracted two times with 100 mL of hexanes. The hexane layers were combined, and the solvent was removed by rotary evaporation to yield 29.7 mg of hexanes soluble material. Water (42 mL) was then added to the MeOH portion and subsequently extracted four times with 100 mL of CHCl3. The CHCl3 layers were combined and the solvent removed by rotary evaporation to yield 526.7 mg of CHCl3soluble material. The CHCl3-soluble material was chromatographed in portions on Sephadex LH-20 (38 cm × 2 cm, 1:1 CHCl3/MeOH) and monitored by TLC (SiO2, 10:1 CHCl3/MeOH). Fractions 8 through 12 (eluting between 98 and 122 mL) contained a mixture of compounds 1 through 3. These fractions were combined and chromatographed with use of centrifugal countercurrent chromatography (7:20:34:20 Hex/CHCl3/MeOH/H2O, normal phase, (15) Peterson, S. W.; Perez, J.; Vega, F. E.; Infante, F. Mycologia 2002, in press. (16) Bugni, T. S.; Abbanat, D.; Bernan, V. S.; Maiese, W. M.; Greenstein, M.; VanWagoner, R. M.; Ireland, C. M. J. Org. Chem. 2000, 65, 7195-7200.
800 rpm, flow 2 mL/min). Final purification of compounds 1 through 3 was performed by using normal phase HPLC (Rainin SiO2 250 mm × 10 mm) with 5% IPA/hexanes and a 40 min linear gradient to 80% IPA. The solvents were removed under reduced pressure to yield 31.0 mg of brocaenol A (1), 13.1 mg of brocaenol B (2), and 3.4 mg of brocaenol C (3). Brocaenol A (1): [R]25D +34 (c 0.008, MeOH); UV (MeOH) λmax (log ) 224 (4.49), 238 (4.52), 322 (3.94); CDMeOH (c 3.14 × 10-4 M) [θ]267 +9888, [θ]304 -1815; IR ν 3481, 2954, 1741, 1648, 1606 cm-1; 1H, 13C, COSY, and HMBC NMR data are listed in Table 1; HRFABMS m/z 335.0751 ([M + H]+ calcd for C16H15O8, 335.0767). Brocaenol B (2): mp 186-190 °C; [R]25D +142 (c 0.009, MeOH); UV (MeOH) λmax (log ) 236 (3.73), 270 (3.36), 316 (3.15); CDMeOH (c 2.89 × 10-3 M) [θ]275 +7665, [θ]314 -1386; IR ν 3416, 2928, 1738, 1698, 1646, 1604 cm-1; 1H, 13C, and HMBC NMR data are listed in Table 2; HRCIMS (CH4) m/z 346.0707 (M+ calcd for C17H14O8, 346.0688). Brocaenol C (3): [R]25D +83 (c 0.0025, MeOH); UV (MeOH) λmax (log ) 224 (4.07), 244 (s) (3.80), 292 (3.82), 308 (s) 3.78; IR ν 3747, 2821, 1750, 1704, 1649, 1625, 1604 cm-1; 1H, 13C, and
HMBC NMR data are listed in Table 2; HRCIMS (isobutane) m/z 333.0607 ([M + H]+ calcd for C16H13O8, 333.0610).
Acknowledgment. This work was supported by NIH grants CA 36622 and CA 67786 (C.M.I.). Funding for the Varian Unity 500-MHz NMR spectrometer was provided through NCI Grant No. 5 P30 CA 42014 and NIH Grant No. 1 S10RR 06262. Mass spectrometry was performed by Elliot M. Rachlin on a Finnigan Mat 95 funded by NSF grant CHE-9002690 and the University of Utah Institutional Funds Committee. Supporting Information Available: Experimental details for the acetylation and MTPA esterifications of compound 1, as well as 1H and 13C NMR spectra for brocaenols A-C (1-3). This material is available free of charge via the Internet at http://pubs.acs.org. JO020597W
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